be achieved by means of site-directed mutagenesis using oligonucleotides in an analogous way to that
described for deletions (Figures 5.17 and 5.18), but in this case the synthetic oligonucleotide primer con-
tains the desired additional nucleotide(s).
5.6.1.3 Replacements. A common type of mutation is that which maintains the same number of
nucleotides but where part of a sequence is replaced. This is particularly useful for single-base alterations
that lead to a change of amino acid codon. Expression of the mutated gene leads to the production of a pro-
tein with a single amino acid alteration (protein engineering). One replacement method is to introduce a
small deletion at a restriction site followed by ligation into the gap of an oligonucleotide duplex of the
same size but of different sequence. A more general approach involves use of a synthetic oligonucleotide
primer in an analogous way to the introduction of deletions and insertions, but with the same number of
nucleotides in the mutant strand as wild type (Figures 5.17 and 5.18).
190 Chapter 5
Figure 5.17 The use of phosphorothioate-modified nucleotides for in vitromutagenesis